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ORIGINAL ARTICLE J Clin Pathol: first published as 10.1136/jcp.2004.024703 on 27 October 2005. Downloaded from PCR based identification and discrimination of agents of and in paraffin wax embedded tissue R Bialek, F Konrad, J Kern, C Aepinus, L Cecenas, G M Gonzalez, G Just-Nu¨bling, B Willinger, E Presterl, C Lass-Flo¨rl, V Rickerts ......

J Clin Pathol 2005;58:1180–1184. doi: 10.1136/jcp.2004.024703

Background: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. Aims: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. Methods: DNA of 52 blinded samples from five different centres was extracted and used as a template in See end of article for two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of authors’ affiliations zygomycetes...... Results: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological Correspondence to: diagnosis of or aspergillosis, respectively. fumigatus DNA was amplified from Dr R Bialek, Institut fu¨r one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no Tropenmedizin, DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax Universita¨tsklinikum embedding. In addition, eight samples from clinically suspected fungal infections (without histopatholo- Tu¨bingen, Keplerstrasse 15, 72074 Tu¨bingen, gical proof) were examined. The two PCR assays detected a concomitant infection with Absidia Germany; ralf.bialek@ corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another med.uni-tuebingen.de two cases. Accepted for publication Conclusions: The two seminested PCR assays described here can support a histopathological diagnosis of 24 March 2005 mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal ...... treatment.

ilamentous fungi of the genus aspergillus and the order We developed a seminested PCR assay targeting the 18S http://jcp.bmj.com/ cause life threatening diseases in immuno- rDNA of zygomycetes, which does not amplify genes of compromised patients and require early and effective aspergillus species, and we modified a PCR assay targeting F 12 antifungal treatment. Different mycotic infections cannot the mitochondrial DNA of Aspergillus spp, which does not be distinguished by clinical and radiological signs, but the amplify DNA from zygomycetes.20–22 The aim of our study was causative fungi show significantly different antifungal to evaluate the identification and discriminatory efficacy of susceptibilities in vitro and in vivo.1 3–6 Identification of the the two PCR assays in paraffin wax embedded tissue sections infecting can be achieved by culture, but yield is low.7–10 originating from different laboratories. Invasive fungal infections are diagnosed most often by on September 30, 2021 by guest. Protected copyright. histopathology without genus or species identification. MATERIALS AND METHODS Forty two paraffin wax blocks and 10 paraffin wax embedded ‘‘Different mycotic infections cannot be distinguished by tissue sections in sterile tubes from 52 patients at risk for clinical and radiological signs, but the causative fungi invasive fungal infections were sent blinded to our laboratory show significantly different antifungal susceptibilities in in Tu¨bingen, Germany. In total, 28 biopsies of paranasal vitro and in vivo’’ sinuses, orbita, nasal mucosa, palate, or facial bones (summarised as sinus biopsies), 15 lung biopsies, five Polymerase chain reaction (PCR) assays have been biopsies of the central nervous system, three liver biopsies, introduced successfully for the identification of fungal and one skin biopsy were screened for fungal DNA. species.11 12 Fungal cultures are identified by targeting multi- Xylene (1 ml) was added to an Eppendorf tube containing copy genes such as ribosomal DNA (rDNA). However, two 5 mm sections of paraffin wax embedded tissue. The amplification and identification of fungal rDNA directly from paraffin wax was removed by incubation and washing with clinical specimens is impaired by similarities between RNA xylene and ethanol.18 DNA was extracted using the QIAamp sequences from pathogenic and contaminating fungi, ani- tissue kit as recommended by the manufacturer (Qiagen, mals, and .13 The increasing size of databases Hilden, Germany), modified by adding three cycles of containing fungal rDNA sequences reveals that some freezing in liquid nitrogen for one minute and boiling for formerly published primers and hybridisation probes can no five minutes to disrupt the fungal cells.16 longer be regarded as species or genus specific.14 15 To circumvent these problems, genes encoding specific proteins can be targeted, but specific proteins are unknown for most Abbreviations: PCR, polymerase chain reaction; rDNA, ribosomal of the numerous species causing mucormycosis.16–19 DNA

www.jclinpath.com Zygomycetes PCR 1181 J Clin Pathol: first published as 10.1136/jcp.2004.024703 on 27 October 2005. Downloaded from 1 10 20 30 40 50 60 70

Absidia corymbifera Rhizomucor pusillus Apophysomyces elegans Saksenaea vasiformis Mucor hiemalis Cunninghamella bertholletiae Syncephalastrum racemosum

71 80 90 100 110 120 130 140

Absidia corymbifera Rhizomucor pusillus Cokeromyces recurvatus Apophysomyces elegans Saksenaea vasiformis Mucor hiemalis Rhizopus oryzae Cunninghamella bertholletiae Syncephalastrum racemosum

141 150 160 170 180

Absidia corymbifera Rhizomucor pusillus Cokeromyces recurvatus Apophysomyces elegans Saksenaea vasiformis Mucor hiemalis Rhizopus oryzae Cunninghamella bertholletiae Syncephalastrum racemosum

Figure 1 Alignment of 18S ribosomal RNA sequences of zygomycetes amplified by inner primer set ZM1 and ZM3 based on a program according to http://jcp.bmj.com/ Corpet (http://prodes.toulouse.inra.fr/multalin/multialin.html).24 The complimentary primer regions are indicated by black bars. Black letters indicate low or missing consensus in contrast to grey letters, which indicate high consensus at a given nucleotide position. Species and GenBank accession numbers used from top to bottom: Absidia corymbifera (AF113407), Rhizomucor pusillus (AF113434), Cokeromyces recurvatus (AF113416), Apophysomyces elegans (AF113412), Saksenaea vasiformis (AF113442), Mucor hiemalis (AF113428), Rhizopus oryzae, now named R arrhizus (AF113440), Cunninghamella bertholletiae (AF113421), and Syncephalastrum racemosum (X89437).

Alignment of 18S rDNA sequences from Rhizopus arrhizus The reaction mixtures of the primary PCRs consisted of (GenBank accession number AF113440) and Aspergillus 10 ml previously extracted DNA in a total volume of 50 ml, on September 30, 2021 by guest. Protected copyright. fumigatus (GenBank AB008401) by special BLAST program with final concentrations of 10mM Tris/HCl (pH 8.3), 50mM (bl2seq) at the National Center for Biotechnology KCl, and 2.5mM MgCl2 (106 Perkin-Elmer buffer II plus Information, Washington, DC, USA, revealed significant MgCl2 solution; Roche Molecular Systems, Branchburg, New differences within the V4 and V5 variable regions.11 Primers Jersey, USA); 1mM of each primer of the outer primer sets within this region show homologies to 18S rDNA of agents of (ZM1/ZM2 and P2/Asp2, respectively; Roth, Karlsruhe, zygomycosis deposited in GenBank.23 The outer primers ZM1 Germany); 1.5 U of AmpliTaq DNA polymerase (Roche); (59-ATT ACC ATG AGC AAA TCA GA-39) and ZM2 (59-TCC and 100mM of each dNTP (Promega, Madison, Wisconsin, GTC AAT TCC TTT AAG TTT C-39) are complementary to USA). Identical reaction mixtures were used for the nested nucleotide positions 711 to 730 and 1117 to 1096 (R arrhizus, PCRs except that 1 ml of the first reaction, 50mM of each AF113440), respectively. Products of the seminested reaction dNTP, and 1mM of each inner primer (ZM1/ZM3 and P1/P2) using primers ZM1 and ZM3 (59-CAA TCC AAG AAT TTC were used. All reaction mixtures were thermally cycled once ACC TCT AG-39) are 175 to 177 bp long, depending on the at 94˚C for five minutes, 35 times at 94˚C for 30 seconds, 50˚C species (fig 1).24 for 30 seconds, and 72˚C for one minute, and then once at To enhance the sensitivity of a published A fumigatus PCR, 72˚C for five minutes. a third primer was added to run a seminested PCR assay.20–22 To validate the presence of amplifiable DNA and absence of P2 (59-CTT TGG TTG CGG GTT TAG GGA TT-39) and Asp2 inhibitory substances a PCR was performed using the primer (59-GGG AGT TCA AAT CTC CCT TGG G-39) are complimen- set G1 (59-GAA GAG CCA AGG ACA GGT AC-39) and G2 (59- tary to nucleotide positions 938–916 and 738–759, respec- CAA CTT CAT CCA CGT TCA CC-39) targeting the human tively, of A fumigatus mitochondrial transfer RNA (GenBank b globin gene (nucleotides 70400–70667; accession number, accession number L37095). The product of the seminested NG_000007.3). The conditions were as described above reaction using P1 (59-GAA AGG TCA GGT GTT CGA GTC AC- except that 5 ml DNA extract and 3mM MgCl2 were used, 39) and P2 is 135 bp long.20 and the extension time was reduced from one minute to 45

www.jclinpath.com 1182 Bialek, Konrad, Kern, et al

seconds. When the result was negative, DNA extraction was DNA was amplified. Nine samples were negative in both PCR J Clin Pathol: first published as 10.1136/jcp.2004.024703 on 27 October 2005. Downloaded from repeated if enough material was available. assays. In four of them no human b globin DNA could be All PCRs were run in a Primus PCR thermocycler Tc 9600 detected by the control PCR. (MWG Biotech, Ebersberg, Germany). PCR products were Aspergillus fumigatus specific DNA was amplified from 11 of analysed by electrophoresis in ethidium bromide stained 17 samples with histologically diagnosed aspergillosis. The 1.8% agarose gels. corresponding zygomycetes PCR assays were negative. No Amplicons of the primary PCRs using DNA extracted from DNA was amplified from the remaining six samples. A laboratory strains of A fumigatus and Absidia corymbifera were concomitant infection was diagnosed in four samples by cloned as described in detail previously.18 Serial dilutions of histopathology but verified by PCR in only one. cloned DNA were used to determine the lower detection limit Another eight samples were obtained from suspected of the nested PCR assays. invasive fungal infections—that is, clinical data were Cloned plasmid DNA (10 ml containing 100 fg (, 5000 ambiguous, fungi were grown from other sites, or the genome equivalents)) was used as a positive control in every histopathological findings were consistent with a fungal PCR assay. Sterile water was included in the DNA extraction infection but not further specified. Identification was after every fifth sample and used in the PCR assays, and accomplished in three samples, and five samples gave reaction mixtures without DNA were run in the first and negative fungal PCR results. nested PCRs to monitor contamination. No cross contamination was seen because all controls The nested PCR products were sequenced by a semiauto- remained negative. A nested PCR product was obtained from mated system (Applied Biosystems Division, Perkin-Elmer all positive controls indicating optimal reaction conditions. Biosystems, Foster City, California, USA) and sequences were A PCR product was detected using a minimum amount of used for a BLAST search of the GenBank database.18 A PCR 0.1 fg plasmid DNA. Assuming up to 40 copies/genome, a assay was considered positive if the product obtained from detection limit of five genome equivalents was calculated for the aspergillus PCR was identical to the sequence in both assays. GenBank, and if the product amplified by zygomycetes PCR showed 100% homology to an 18S rDNA sequence of a DISCUSSION zygomycete. In 25 of 40 samples with amplifiable human DNA, results Results of PCR assays not meeting these criteria were obtained by the two PCR assays were in accordance with the recorded as negative. histopathological findings, and the aetiological agent was The diagnoses and results of microscopy were unblinded identified to the genus or species level. In three additional for further analysis. samples, specific DNA was identified. Hayden et al examined biopsies from tissues with culture confirmed zygomycosis (13 RESULTS samples) and aspergillosis (37 samples), by in situ hybridisa- The aetiological agent was identified by PCR in 18 of the 24 tion using five nucleotide probes.25 According to these biopsies with amplifiable human DNA from the sinus, orbita, authors, several cases of zygomycosis were not interpretable, palate, or other site related to the nasal cavities, in six of the and species identification was not possible. Because of 15 lung biopsies, in all five brain biopsies, in one of the three similarities between fungal ribosomal genes, species identi- liver biopsies, and in the one skin biopsy. Seventeen of 48 fication can only be accomplished by sequencing the PCR biopsies with amplifiable human DNA remained negative by product. However, even targets of 280 bp within the 18S both fungal PCR assays. rDNA might not be sufficient to discriminate between http://jcp.bmj.com/ As shown in table 1, a genus or species identification was genera.26 Immunohistochemistry has been used to identify achieved in 14 of 23 samples diagnosed as zygomycosis by and discriminate aspergillosis and mucormycosis, in addition histopathology. The corresponding aspergillus PCR assays to candidosis, scedosporiosis, and fusariosis, but species were negative. PCR products of Rhizomucor pusillus and identification of zygomycetes was not achieved.27–29 R miehei are identical using the primers described in this Agents of mucormycosis and aspergillosis are considered study, and recorded as Rhizomucor spp. In one sample ubiquitous fungi that might be found in nasal mucous.30 zygomycetes PCR was negative, but A fumigatus specific Although we mainly examined biopsies from airways, only a on September 30, 2021 by guest. Protected copyright.

Table 1 Results of PCR assays correlated to the type of biopsy tissue and histopathological diagnosis

Tissue and number of PCR results Total

Diagnosis by histopathology PCR results and identified species Sinus* Lung Brain Liver Skin

Zygomycosis (n = 23) Rhizomucor spp 511–18 Mucor hiemalis 2––––2 Absidia corymbifera 11–––2 Rhizopus arrhizus ––1––1 Aspergillus fumigatus 1––––1 Negative (including 4 b globin PCR 9––––9 negative DNA extracts) Aspergillosis (n = 17) Aspergillus fumigatus 632––11 Negative – 6 – – – 6 Mixed infection (n = 4) Rhizomucor spp and A fumigatus 1––––1 Rhizomucor spp 2 – – – – 2 Negative 1 – – – – 1 Suspected invasive fungal infection (n = 8) Rhizopus arrhizus and A fumigatus ––1––1 Absidia corymbifera –––1–1 A fumigatus –1–––1 Negative – 3 – 2 – 5 Total 28 15 5 3 1 52

*Biopsies of paranasal sinuses, nasal mucosa, orbita, palate, or facial bones; Rhizomucor pusillus and R miehei were not distinguishable by sequencing, and results were reported as Rhizomucor spp.

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limited number of samples were identified with DNA from used for epidemiological and diagnostic purposes, and to J Clin Pathol: first published as 10.1136/jcp.2004.024703 on 27 October 2005. Downloaded from fungi not detected by histopathology. Aspergillus fumigatus guide antifungal treatment. specific DNA was identified from only one case of histo- pathological diagnosis of zygomycosis; this might have been ACKNOWLEDGEMENTS the result of contamination of the sinuses. However, a We thank Dr H Mair, Department of Pathology, University misdiagnosis by histopathology cannot be excluded; this has Innsbruck, Dr R Sedivy, Department of Clinical Pathology, been described even in culture confirmed cases.25 The University Vienna, Dr G Tucek, Department of Pathology, Kaiser numerous biopsies in our study with only one type of fungal Franz Josef Krankenhaus, Vienna, and J Beck-Managetta, Department of Oral and Maxillo-Facial Surgery, DNA identified by PCR or with negative results by both PCR Landeskrankenhaus Salzburg, Austria for providing paraffin wax assays argue against a high rate of contamination of the embedded tissue samples. The authors wish to thank Dr JM Scotter biopsies or during paraffin wax embedding. Specimens from for her thoughtful review of the manuscript. culture confirmed cases are necessary to determine the specificity and sensitivity of the PCR assays...... Authors’ affiliations R Bialek, F Konrad, J Kern, Institute for Tropical Medicine, University ‘‘Because of similarities between fungal ribosomal genes, Hospital Tu¨bingen, Keplerstrasse 15, 72074 Tu¨bingen, Germany species identification can only be accomplished by C Aepinus, Institute for Medical , Virology and Hygiene, sequencing the polymerase chain reaction product’’ University Hospital Rostock, Rostock D-18057, Germany L Cecenas, Departamento de Pathologia, Universidad Autonoma Nuevo No specific fungal DNA was amplified from 16 of 44 Leon, Monterrey, NL, Mexico CP 64460 biopsies with fungal infection identified by histopathology. G M Gonzalez, Departamento de Microbiologia, Universidad The failure to amplify specific DNA might result from fungal Autonoma Nuevo G Just-Nu¨bling, V Rickerts, Medizinische Klinik III, University Hospital, DNA concentrations below detection limits, a focal infection D-60590 Frankfurt/Main, Germany with varying amounts of fungal elements within the tissue, B Willinger, Monterrey, Mexico, and Institute of Hygiene and Medical or hyalohyphomycosis caused by or Microbiology, Medical University of Vienna, A-1090 Wein, Austria species. According to Bretagne et al, DNA of several E Presterl, Department of Medicine, Division of Infectious Diseases and aspergillus species will be amplified by the primers, but Hospital , CH-8091 Zurich, Switzerland sequences for comparison are not deposited in GenBank.20 In C Lass-Flo¨rl, Department of Hygiene and Social Medicine, University of four cases, no amplifiable DNA was detected, probably Innsbruck, A-6010 Innsbruck, Austria 31 because of the destruction of DNA during formalin fixation. The data presented here are part of the doctoral thesis of F Konrad. A dual infection diagnosed by histopathology was detected by PCR in only one of four cases, but also in a biopsy of suspected fungal infection. There are few reports on REFERENCES concomitant fungal infections.32 Because of the differences 1 Husain S, Alexander BD, Munoz P, et al. Opportunistic mycelial fungal in antifungal susceptibilities, the identification of both agents infections in organ transplant recipients: emerging importance of non- aspergillus mycelial fungi. Clin Infect Dis 2003;37:221–9. is mandatory to institute appropriate antifungal treatment. 2 Marr KA, Carter RA, Crippa F, et al. Epidemiology and outcome of mould Histopathology appears to be the most sensitive method for infections in hematopoietic stem cell transplant recipients. Clin Infect Dis the detection of invasive fungal infection.7 The two PCR 2002;34:909–17. assays described here can be used for individual cases, for 3 Diekema DJ, Messer SA, Hollis RJ, et al. Activities of caspofungin,

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